Posttranscriptional regulation of Dmrt1 during sexual development in Medaka

• Applicants: Dr. Amaury Herpin; Professor Manfred Schartl, Ph.D.
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 264892131

The development of males and females and bisexual reproduction is the most prevalent mode in metazoans. Despite the generality of the phenomenon itself the mechanisms how sex is determined are very different among various groups and have evolved repeatedly and independently. The underlying molecular pathways can change quickly during evolution, even within closely related groups of organisms. The factors involved in sex determination and gonad differentiation can also be substantially different. A single exception is the transcription factor Dmrt1, which is highly conserved in all metazoans and has been shown to be involved in sexual development from flies and worms up to humans. In vertebrates a precise regulation of gene expression with respect to cell type specificity, time of development and transcript level is requested to ensure a proper function in sex determination. We are using the small aquarium fish model medaka (Oryzias latipes) to study the role of Dmrt1 in vertebrate sexual development and on a more general level to contribute to an understanding of the evolution of the high plasticity of sex determining mechanisms. We have shown earlier that in medaka a duplicated copy of dmrt1 (dmrt1bY) is the master male sex-determining gene. We found that regulation of dmrt1bY expression most critically occurs on the posttranscriptional level through a cell type specific and developmental time dependent differential mRNA stability. We identified a protein-binding motif in the 3' UTR of dmrt1bY to be responsible for this regulation. Interestingly, this motif is highly conserved in dmrt1 homologues of invertebrates and vertebrates, including human. In this project we want to identify the proteins which interact with the dmrt1 3' UTR and regulate its expression, as well as their interaction with other germline and gonad determining genes. On the one hand, we propose to study two candidate proteins that have been reported to bind to two different sequences, which both together make up the dmrt1 3' UTR consensus binding motif. This will be done after recombinant expression of both proteins and assaying their binding activities in vitro. In parallel, we will take an unbiased approach and screen for binding factors by affinity column purification. For functional characterization, we will study the expression pattern of the 3' UTR box binding proteins and further on use factor overexpression, gene knockdown and gene knockout approaches to validate their role in dmrt1 posttranscriptional expression regulation. Finally, the factors characterized in the fish model will be studied in mouse for their role in sexual development. This should contribute to a better understanding how the highly conserved sex determining factor Dmrt1 exerts its function in vertebrates and increase the knowledge about the complex regulatory interactions that lead to determination of the gonad anlage towards testis or ovary development in vertebrates.

• DFG Programme: Research Grants